[[Image:TUDelft_Alkane_degradation_route.png|450px|thumb|right|Figure 1: Schematic description of the alkane degradation pathway with the corresponding genes.]]

===Introduction===

===Introduction===

The Alkanivore ''E.coli'' strain has been designed to carry the genes required for the conversion of medium (C<sub>5</sub>-C<sub>13</sub>) and long chain (C<sub>15</sub>-C<sub>36</sub>) alkanes. A general scheme for the oxidation of the alkanes is illustrated in figure 1.

The Alkanivore ''E.coli'' strain has been designed to carry the genes required for the conversion of medium (C<sub>5</sub>-C<sub>13</sub>) and long chain (C<sub>15</sub>-C<sub>36</sub>) alkanes. A general scheme for the oxidation of the alkanes is illustrated in figure 1.

To create the alkane degradation constructs a number of genes encoding for alkane degradation enzymes were synthesized and combined with promoters and ribosome binding sites obtained from the BioBrick distribution plates. Combination of these genes resulted in the following BioBrick constructs (the intermediates have also been submitted to the registry).

To create the alkane degradation constructs a number of genes encoding for alkane degradation enzymes were synthesized and combined with promoters and ribosome binding sites obtained from the BioBrick distribution plates. Combination of these genes resulted in the following BioBrick constructs (the intermediates have also been submitted to the registry).

This system facilitates the initial oxidation step of C5-C13 alkanes along with that of C5-C8 cycloalkanes towards their respective alcohols. Based on the literature on this topic [ref] it is expected that the in-house mechanism of E.coli will be able to further degrade the products of this pathway.

This system facilitates the initial oxidation step of C<sub>5</sub>-C<sub>13</sub> alkanes along with that of C<sub>5</sub>-C<sub>8</sub> cycloalkanes towards their respective alcohols. Based on the literature on this topic [5] it is expected that the in-house mechanism of ''E.coli'' will be able to further degrade the products of this pathway.

The AH construct consists of the sequences encoding for:

The AH construct consists of the sequences encoding for:

-

*alkane 1-monooxygenase (alkB2); an integral cytoplasmic membrane monooxygenase of which homologs have been reported for varying genus and species. This is the catalytic component of the AH system and as such oxidizes (cyclo)alkanes to their respective (cyclo)alkanols by transferring one oxygen atom from molecular oxygen to the alkane.

+

*Alkane 1-monooxygenase (alkB2); an integral cytoplasmic membrane monooxygenase of which homologs have been reported for varying genus and species. This is the catalytic component of the AH system and as such oxidizes (cyclo)alkanes to their respective (cyclo)alkanols by transferring one oxygen atom from molecular oxygen to the alkane.

-

*Rubredoxin reductase (rubR); catalyzes the reduction of the second oxygen atom released from molecular oxygen using electrons supplied by NADH.

+

*Rubredoxin reductase (rubB); catalyzes the reduction of the second oxygen atom released from molecular oxygen using electrons supplied by NADH.

*Rubredoxin (rubA3); facilitates the transfer of electrons from NADH to rubredoxin reductase.

*Rubredoxin (rubA3); facilitates the transfer of electrons from NADH to rubredoxin reductase.

*Rubredoxin (rubA4); an electron-carrier protein required by the AH system.

*Rubredoxin (rubA4); an electron-carrier protein required by the AH system.

-

The AH construct was designed to harbour all four of the required coding sequences, each behind its own RBS region and sharing a constitutive promoter. For more information on part specifics you may view this part in the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398014 '''parts registry''']

+

The AH construct was designed to harbor all four of the required coding sequences -each behind its own RBS region- sharing a constitutive promoter.

+

+

View this part in the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398014 '''parts registry''']

[[Image:BBa_K398014_AlkB2_RubA3_RubA4_RubR.png|350px]]

[[Image:BBa_K398014_AlkB2_RubA3_RubA4_RubR.png|350px]]

-

===BBa_K398017 - Long-chain (C15-36) alkane conversion===

+

===BBa_K398017 - Long-chain (C15-C36) alkane conversion (ladA)===

-

For the first step in the long-chain alkane degradation pathway the apoprotein ladA was implemented [2]; an alkane monooxygenase from ‘’Geobacillus thermodenitrificans’’ NG80-2. This protein facilitates the conversion of long-chain alkanes (from C15 up to at least C36) to their corresponding primary alcohols in the presence of flavin mononucleotide, oxygen and NADH.

+

For the first step in the long-chain alkane degradation pathway ladA was implemented [2]; a flavoprotein alkane monooxygenase native to ''Geobacillus thermodinitrificans NG-80-2''. It has been found to specifically oxidize the terminal regions of alkanes ranging from C<sub>15</sub> up to at least C<sub>36</sub>. The product is the corresponding primary alkanol. LadA forms a catalytic complex with flavin mononucleotide (FMN) which utilizes dioxygen to insert an oxygen atom into the substrate.

+

+

The general catalytic function involves three chemical processes:

+

*Reduction of the cofactor flavin mononucleotide (FMN to FMNH2) by NAD(P)H

+

*Reaction of FMNH2 with O2

+

*Binding, orienting, and activating the substrate for its oxygenation

+

+

LadA's ability to preferentially capture long-chain alkanes for oxidation sets it apart from other flavoprotein monooxygenases.

View this part in the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398017 '''parts registry''']

View this part in the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398017 '''parts registry''']

Line 32:

Line 46:

[[Image:TUDelft_BBa_K398017.png|150px]]

[[Image:TUDelft_BBa_K398017.png|150px]]

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===BBa_K398018 - Long-chain alkanol conversion===

+

===BBa_K398018 - Medium-chain alkanol conversion (ADH)===

-

The next step is catalyzed by a zinc-independent alcohol dehydrogenase from Bacillus thermoleovorans B23; a thermophilic bacterium [3-4]. The enzyme converts long-chain alkanols into their respective alkanals by oxidation of NADH.

+

The following step in the oxidation pathway is catalyzed by a zinc-independent alcohol dehydrogenase from ''Geobacillus thermoleovorans B23''; a thermophilic bacterium [3]. The enzyme converts medium-chain alkanols into their respective alkanals by reduction of NAD into NADH.

View this part in the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398018 '''parts registry''']

View this part in the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398018 '''parts registry''']

For the final step in the medium-chain oxidation the aldehyde dehydrogenase from the thermophile ''Geobacillus thermoleovorans B23'' is implemented. It functions as an octamer, requiring NAD+ as coenzyme. The optimum condition for activity lies at temperatures between 50 and 55 degrees Celsius and a pH of 10 [4].

Latest revision as of 22:21, 27 October 2010

Alkane Degradation Parts

Figure 1: Schematic description of the alkane degradation pathway with the corresponding genes.

Introduction

The Alkanivore E.coli strain has been designed to carry the genes required for the conversion of medium (C5-C13) and long chain (C15-C36) alkanes. A general scheme for the oxidation of the alkanes is illustrated in figure 1.

To create the alkane degradation constructs a number of genes encoding for alkane degradation enzymes were synthesized and combined with promoters and ribosome binding sites obtained from the BioBrick distribution plates. Combination of these genes resulted in the following BioBrick constructs (the intermediates have also been submitted to the registry).

BBa_K398014 - Medium-chain (C5-C13) alkane conversion (alkB)

Our medium-chain hydrocarbon degrading strain contains the alkane hydroxylase (AH) native to Gordonia sp. TF6 [1].
This system facilitates the initial oxidation step of C5-C13 alkanes along with that of C5-C8 cycloalkanes towards their respective alcohols. Based on the literature on this topic [5] it is expected that the in-house mechanism of E.coli will be able to further degrade the products of this pathway.

The AH construct consists of the sequences encoding for:

Alkane 1-monooxygenase (alkB2); an integral cytoplasmic membrane monooxygenase of which homologs have been reported for varying genus and species. This is the catalytic component of the AH system and as such oxidizes (cyclo)alkanes to their respective (cyclo)alkanols by transferring one oxygen atom from molecular oxygen to the alkane.

Rubredoxin reductase (rubB); catalyzes the reduction of the second oxygen atom released from molecular oxygen using electrons supplied by NADH.

Rubredoxin (rubA3); facilitates the transfer of electrons from NADH to rubredoxin reductase.

Rubredoxin (rubA4); an electron-carrier protein required by the AH system.

The AH construct was designed to harbor all four of the required coding sequences -each behind its own RBS region- sharing a constitutive promoter.

BBa_K398017 - Long-chain (C15-C36) alkane conversion (ladA)

For the first step in the long-chain alkane degradation pathway ladA was implemented [2]; a flavoprotein alkane monooxygenase native to Geobacillus thermodinitrificans NG-80-2. It has been found to specifically oxidize the terminal regions of alkanes ranging from C15 up to at least C36. The product is the corresponding primary alkanol. LadA forms a catalytic complex with flavin mononucleotide (FMN) which utilizes dioxygen to insert an oxygen atom into the substrate.

The general catalytic function involves three chemical processes:

Reduction of the cofactor flavin mononucleotide (FMN to FMNH2) by NAD(P)H

Reaction of FMNH2 with O2

Binding, orienting, and activating the substrate for its oxygenation

LadA's ability to preferentially capture long-chain alkanes for oxidation sets it apart from other flavoprotein monooxygenases.

BBa_K398018 - Medium-chain alkanol conversion (ADH)

The following step in the oxidation pathway is catalyzed by a zinc-independent alcohol dehydrogenase from Geobacillus thermoleovorans B23; a thermophilic bacterium [3]. The enzyme converts medium-chain alkanols into their respective alkanals by reduction of NAD into NADH.

BBa_K398029 and BBa_K398030 - Medium-chain alkanal conversion (ALDH)

For the final step in the medium-chain oxidation the aldehyde dehydrogenase from the thermophile Geobacillus thermoleovorans B23 is implemented. It functions as an octamer, requiring NAD+ as coenzyme. The optimum condition for activity lies at temperatures between 50 and 55 degrees Celsius and a pH of 10 [4].